A R2R3-MYB gene-based marker for the non-darkening seed coat trait in pinto and cranberry beans (Phaseolus vulgaris L.) derived from 'Wit-rood boontje'

Abstract

The gene Phvul.010G130600 which codes for a MYB was shown to be tightly associated with seed coat darkening in Phaseolus vulgaris and a single nucleotide deletion in the allele in Wit-rood disrupts a transcription activation region that likely prevents its functioning in this non-darkening genotype. The beige and white background colors of the seed coats of conventional pinto and cranberry beans turn brown through a process known as postharvest darkening (PHD). Seed coat PHD is attributed to proanthocyanidin accumulation and its subsequent oxidation in the seed coat. The J gene is an uncharacterized classical genetic locus known to be responsible for PHD in common bean (P. vulgaris) and individuals that are homozygous for its recessive allele have a non-darkening (ND) seed coat phenotype. A previous study identified a major colorimetrically determined QTL for seed coat color on chromosome 10 that was associated with the ND trait. The objectives of this study were to identify a gene associated with seed coat postharvest darkening in common bean and understand its function in promoting seed coat darkening. Amplicon sequencing of 21 candidate genes underlying the QTL associated with the ND trait revealed a single nucleotide deletion (c.703delG) in the candidate gene Phvul.010G130600 in non-darkening recombinant inbred lines derived from crosses between ND 'Wit-rood boontje' and a regular darkening pinto genotype. In silico analysis indicated that Phvul.010G130600 encodes a protein with strong amino acid sequence identity (70%) with a R2R3-MYB-type transcription factor MtPAR, which has been shown to regulate proanthocyanidin biosynthesis in Medicago truncatula seed coat tissue. The deletion in the 'Wit-rood boontje' allele of Phvul.010G130600 likely causes a translational frame shift that disrupts the function of a transcriptional activation domain contained in the C-terminus of the R2R3-MYB. A gene-based dominant marker was developed for the dominant allele of Phvul.010G130600 which can be used for marker-assisted selection of ND beans.

Fig. 1

Sequence alignment of the gene Phvul.010G130600 in ‘Wit-rood boontje’ [parent with the non-darkening (ND) seed coat phenotype] with 1533-15 [parent with the slow darkening (SD) seed coat phenotype]. Wit-rood boontje displayed a single nucleotide deletion (c.703delG, in red box) in the 3rd exon region (yellow shaded). The green-shaded segment identifies the 5ʹ untranslated region (UTR), the blue-shaded segment identifies the 3ʹ UTR, and the lowercase letters identify the introns (color figure online)

Fig. 2

Sequence alignments of genomic segments six different genotypes corresponding to the exon region (yellow shaded) of gene Phvul.010G130600 containing the single nucleotide polymorphism (c.703delG, bordered by red lines) associated with the non-darkening trait in ‘Wit-rood boontje.’ The sequence in Wit-rood boontje [parent A with the non-darkening (ND) seed coat phenotype] is compared to: RIL29 (ND), 1533-15 [parent B with the slow darkening (SD) seed coat phenotype], RIL81 [regular darkening (RD)], Othello (RD pinto bean variety), Etna (RD cranberry variety), and G19833 (P. vulgaris reference genome G19833) (color figure online)

Fig. 3

Multiple sequence alignment of R2R3-MYB proteins and putative R2R3-MYB proteins encoded by the gene homologs of Phvul.010G130600 in P. vulgaris G19833 showing characteristic motifs. The alignment includes putative translations of homologs of Phvul.010G130600: in ‘Wit-rood-boontje’ [parent A with the non-darkening (ND) seed coat phenotype], 1533-15 [parent B with slow darkening (SD) seed coat phenotype], RIL81 [regular darkening (RD) line], RIL29 (ND line), MtPAR (Medtr8G020490 in M. truncatula), GmMYB6 (Glyma.16G007100 in Glycine max), GmMYB8 (Glyma.07G037700 in Glycine max), CcMYB8 (LOC109802394 in Cajanus cajan), MpC1 (C1 in Mucuna pruriens), and VrWER-Like (LOC106773092 in Vigna radiata). Motifs characteristic of MYB are identified, including the primary structure R2 and R3 DNA-binding domains [-W-(X19)-W-(X19)-W-] and [-F/I- (X18)-W-(X18)-W-]; in which W represents tryptophan and X represents any amino acid. [D/E]Lx₂[R/K]x₃Lx₆Lx₃R is the bHLH-interaction motif located on helices 1 and 2 of the R3 repeat; The EDLL motif and amino acid residues in the transactivation domain are shown, containing characteristic E (glutamic acid), D (aspartic acid), and L (leucine) amino acids

Fig. 4

Neighbor-joining cluster analysis of putative R2R3-MYB proteins encoded by gene homologs of Phvul.010G130600 in P. vulgaris G19833. The cluster includes the Phvul.010G130600 protein sequence: in 1533-15 [parent B with the slow darkening (SD) seed coat phenotype] and RIL81 (SD line), the Phvul.010G130600 protein sequence: in ‘Wit-rood-boontje’ [parent A with the non-darkening (ND) seed coat phenotype] and RIL29 (ND line), LOC106773092 transcription factor WER-like in Vigna radiata, MpC1 anthocyanin regulatory C1 protein in Mucuna pruriens, LOC109802394 transcription factor MYB8 in Cajanus cajan, Glyma.16G007100 transcription repressor MYB6 isoform X1 and Glyma.07G037700 transcription factor MYB8 in Glycine max, and Medtr8G020490 MYB transcription factor MtPAR in M. truncatula

Fig. 5

Sequence alignment of an R2R3-MYB protein encoded by the gene Phvul.010G130600 of the reference genome P. vulgaris G19833 reference genome, ‘Wit-rood-boontje’ (parent A with the non-darkening (ND) seed coat phenotype), and 1533-15 (parent B with slow darkening (SD) seed coat phenotype). [-W-(X19)-W-(X19)-W-] and [-F/I- (X18)-W-(X18)-W-] are the primary structure of R2 and R3 DNA-binding domains, in which W represents tryptophan and X represents any amino acid. [D/E]Lx₂[R/K]x₃Lx₆Lx₃R is the bHLH-interaction motif located on helices 1 and 2 of the R3 repeat. E (glutamic acid), D (aspartic acid), and L (leucine) amino acids in the transactivation domain represent the EDLL motif and negatively charged residues

Fig. 6

Phvul.010G130600 SNP-based marker and the marker screen of regular, slow (SD) and non-darkening (ND) recombinant inbred lines (RIL), breeding lines and varieties of bean. a PCR binding sites for PCR primers (Table 3) in regular darkening (RD) and non-darkening (ND) genotypes. The SNP is identified with a red box. b PCRs with genomic DNA from a ND genotype like ‘Wit-rood boontje’ did not produce an amplicon. PCRs with genomic DNA from a slow darkening (SD) parent 1533-15 produced a single DNA fragment with a size of 254 bp. c RILs88-101 are recombinant inbred lines from a ND ‘Wit-rood-boontje’ x SD 1533-15 cross. d Burdett, Othello, Windbreaker, Sequoia, PT11-9, Kodiak, Kimberly, La Paz, 16-NDP1, UI 114, and ME105 are Pinto bean varieties and lines. Etna, CBX 1148, C15HR009, Red Rider, OAC Racer, C13HR185, and 8184 (94CTCOOP-8184) are cranberry bean varieties and lines. Labels that are highlighted red, yellow, and blue represent seeds with (regular darkening) RD, SD, and ND phenotypes, respectively (color figure online)